The use of identical particles for pre-treatment and treatment procedures may improve the predictive value of pre-treatment analysis of distribution. The aim of this study was to analyze the agreement between 166Ho-scout and 166Ho-therapeutic dose in comparison with the agreement between 99mTc-MAA and 166Ho-therapeutic dose.As an alternative to technetium-99m-macroaggregated albumin (99mTc-MAA and 166Ho-scout) before treatment in 53 patients. First, qualitative assessment was performed by two blinded nuclear medicine physicians who visually rated the agreement between the 99mTc-MAA, 166Ho-scout, and 166Ho-therapeutic dose SPECT-scans  on a 5-point scale. Second, agreement was measured quantitatively by delineating lesions and normal liver on FDG-PET/CT. These volumes of interest (VOIs) were co-registered to the SPECT/CT images. The predicted absorbed doses (based on 99mTc-MAA and 166Ho-scout) were compared with the actual absorbed dose on post-treatment SPECT.Two separate scout dose procedures were performed (166Ho-scout was superior with a median score of 4 vs. 2.5 for 99mTc-MAA (p < 0.001). The quantitative analysis showed significantly narrower 95%-limits of agreement for 166Ho-scout in comparison with 99mTc-MAA when evaluating lesion absorbed dose . Evaluation of normal liver absorbed dose did not show difference in agreement between both scout doses and 166Ho-therapeutic dose .A total of 23 procedures  were included for analysis. In the qualitative analysis, 166Ho-scout was shown to have a superior predictive value for intrahepatic distribution in comparison with 99mTc-MAA.In this study,  Aside from predicting the lung shunt and (other) non-target embolization, 99mTc-MAA is also used to predict the activity distribution in the liver. Several studies have shown that the value of 99mTc-MAA to predict the distribution of 90Y in the liver is limited [99mTc-MAA differ greatly from 90Y-microspheres. This may explain the difference in biodistributions.Treatment planning for radioembolization  of liver tumors is generally performed with technetium-99m macroaggregated albumin particles ( limited –4. Shape90Y)-microspheres, microspheres containing the radioactive element holmium-166 (166Ho) have recently become available . The radionuclide 166Ho provides several advantages over 90Y with regard to imaging possibilities. The element holmium itself is paramagnetic and can therefore be visualized—and quantified—on MRI [166Ho emits low-energy gamma radiation, which can be used for quantitative SPECT/CT imaging [166Ho-microspheres (consisting of approximately 3 million microspheres with an activity of 250 MBq) can be used for treatment planning instead of 99mTc-MAA [166Ho-microspheres for both procedures. The aim of the current study was to analyze whether the intrahepatic distribution of 166Ho-scout has a better agreement with the 166Ho-therapeutic dose distribution in comparison with 99mTc-MAA.As an alternative to the yttrium-90  studies were analyzed . All patients had unresectable liver metastases from various primaries. The institutional review board approved these studies and all patients provided written informed consent. All patients received treatment planning with the conventional 99mTc-MAA and subsequently with a scout dose of 250 MBq of 166Ho-microspheres . Patients were included for analysis if they had received both pre-treatment administrations of 99mTc-MAA and 166Ho-scout before treatment with 166Ho-microspheres.Patients treated with 99mTc-MAA  was injected. 99mTc-MAA was injected slowly at approximately 5 ml/min. All injections (both 99mTc-MAA and 166Ho-microspheres) were performed with a standard 2.4F or 2.7F microcatheter . At the end of the procedure, the access site in the groin was compressed for hemostasis. 99mTc-MAA injection was followed by planar- and SPECT/CT imaging to check for extrahepatic deposition and excessive lung shunting (> 30 Gy lung absorbed dose). If no contra-indications were found, the patient was scheduled for treatment. In case the 99mTc-MAA procedure had to be repeated because of extrahepatic activity, only the most recent 99mTc-MAA data were used for analysis. No vessels were coil-embolized after the final 99mTc-MAA procedures. On the day of treatment, patients underwent a second treatment-planning procedure in the morning. During this procedure, the injection position(s) from the first treatment planning procedure was mimicked and patients received a scout dose of 250 MBq of 166Ho-microspheres (approximately 3 million microspheres). At the end of the procedure, the catheter and microcatheter were removed. The vascular sheath was left in the groin in order to secure vascular access for the treatment procedure in the afternoon. The sheath was connected to a pressurized bag of saline (for continuous flushing) and secured with sterile tape. This second treatment-planning procedure was again followed by planar imaging and SPECT/CT. If there were no contra-indications for SIRT, patients returned to the angio-suite in the afternoon to receive treatment with 166Ho-microspheres. Catheters were introduced via the vascular sheath that had remained in situ. Again, injection position(s) of the treatment-planning procedures were mimicked. Total treatment activity was planned based on an aimed whole liver absorbed dose of 20, 40, 60, and 80 Gy for patients who were treated in the phase 1 HEPAR study and 60 Gy for patients in the phase 2 HEPAR study [All radioembolization procedures were performed according to the HEPAR study protocol . Non-tarAR study , 11. The99mTc-MAA, 166Ho-scout, and 166Ho-therapeutic dose) was retrospectively analyzed by three observers . These observers independently reviewed the agreement of all injection positions per procedure on digital subtraction angiography images. Agreement was rated on a 4-point scale . Only the patients with good or very good agreement  between all the injection positions were included in this study.In order to exclude cases in which differences in injection positions might have influenced distribution, agreement of catheter-tip positions between the three procedures . Agreement between the procedures was checked based on the administered activity per injection position, which was corrected for any residual activity. A maximum deviation of 10% between the two pre-treatment and post-treatment injections was accepted.99mTc-MAA SPECT images were acquired on a 512 × 512 matrix (15 procedures) and a 128 × 128 matrix (8 procedures). An energy window of 129.1- to 150.5-keV and a low-energy general-purpose collimator were used. Pre- and post-treatment 166Ho SPECT images  were acquired on a 128 × 128 matrix (22 procedures) and a 512 × 512 matrix (1 procedure) in combination with a 74.9- to 87.1-keV energy window and a medium-energy general-purpose collimator. Imaging was performed with 120 projections over a noncircular orbit of 360° (99mTc-MAA: 30s/projection (Philips) or 20s/projection (Siemens) and 166Ho; 30s/projection). An in-house developed and validated Monte-Carlo-based reconstruction algorithm (Utrecht Monte-Carlo System (UMCS)) intrinsically correcting for attenuation and scatter was used for the reconstruction of the 99mTc-MAA and 166Ho HEPAR I data [99mTc-MAA; 6 iterations, 8 subsets, 166Ho; 5 iterations 8 subsets). The scatter correction for the HEPAR II 99mTc-MAA data was applied by using the dual energy window method with two adjacent energy windows (both 15% width) and a lower window weight of 0.5. The HEPAR II 166Ho data was reconstructed without scatter correction. For 99mTc-MAA data, a Gaussian post-reconstruction filter of 5 mm in full width at half maximum was also included. Reconstructed voxel sizes were 4.66 × 4.66 × 4.66 mm3 for all Philips images and in the range of 0.70 × 0.70 × 4.03 mm3 to 3.90 × 3.90 × 4.03 mm3 for the Siemens 99mTc-MAA and 4.80 × 4.80 × 4.80 mm3 for the Siemens 166Ho images, respectively.Pre- and posttreatment SPECT/CT imaging was performed on a dual-headed gamma camera  and a SPECT/CT camera . In all cases, the same scanner was used for pre- and posttreatment SPECT/CT. Pre-treatment 99mTc-MAA and 166Ho-scout with the post-treatment 166Ho-therapeutic dose. Image sets of all three SPECT scans were coupled, blinded, and simultaneously presented to the reviewers in our Picture Archive and Communication System . The post-treatment SPECT was marked “post-treatment” to allow the reviewers to compare it with the other two pre-treatment SPECT scans (which were marked “pre-treatment”). No information was provided regarding the type of pre-treatment scan. To prevent bias, both pre-treatment scans were placed randomly on the screen for each patient. Visual agreement was rated on a 5-point scale .Two nuclear medicine physicians independently compared the intrahepatic distribution of activity of 99mTc-MAA SPECT, 166Ho-scout SPECT, and 166Ho-therapeutic dose SPECT images  for each VOI, based on both pre-treatment SPECT images, in comparison with an actual amount of 166Ho based on post-treatment SPECT. The lesion-absorbed dose was calculated by dividing the activity measured in the lesion VOI plus the 1-cm margin by the volume of that same lesion VOI without the 1-cm margin. Normal liver activity was calculated by subtraction of the measured activity of all lesion VOIs within one patient  without 1-cm margin from the activity of the liver VOI with 1-cm margin. The volume of the normal liver compartment was calculated by subtraction of the total volume of the lesion VOIs without margin from the volume of the liver without margin. Finally, the calculated activity was divided by this volume to calculate the normal liver absorbed dose. No extrahepatic activity distribution and no interval change in liver morphology were assumed. Contribution of gamma radiation was neglected in calculating the absorbed dose. To illustrate clinical implications, the calculated activity concentrations were converted into units of Gray for both the lesion and normal liver segmentations, using a conversion factor of 15.78 ∙ 10−3 Gy/(MBq/kg) assuming a liver density of 1.06 kg/L [Agreement of intrahepatic distribution was also measured quantitatively. FDG-PET/CT pre-treatment scans were used for segmentation of lesion and liver volumes . In-house-developed software was used for this purpose  . Delinea166Ho treatment scan. The Weighted Kappa (squared) was calculated to classify the interrater agreement [Statistical data analysis was performed using a commercial statistical software package . For the qualitative analysis, medians and interquartile ranges were calculated and the Wilcoxon signed rank test was used to analyze differences in overall agreement scores between both pre-treatment scans and the greement , 15.n), the standard deviation of the differences (s), and the degrees of freedom (n − 1). In this estimation, no corrections were made for any correlation between analyzed lesions within a patient. The 95% confidence intervals were calculated by the multiplication of the standard error . Thirty-two procedures were excluded from analysis: in 13 procedures, a mismatch in catheter tip positioning was found; in 10 procedures, data was not completely available (one or more missing angiographic images (n = 4); no pre-treatment FDG-PET/CT (n = 2); no post-treatment SPECT/CT (n = 2); 166Ho-scout was not administered (n = 2)), and in nine procedures, the ratio of activity between the injection positions was not similar. A total of 23 procedures in 22 patients were included for analysis .Fifty-three patients underwent 55 treatment procedures between December 2009 and March 2015 (two patients underwent separate lobar procedures) . In 15 of the 23 procedures, overall agreement between 166Ho-scout and 166Ho-therapeutic dose was rated higher than 99mTc-MAA  and 166Ho-scout (− 108.3 and − 72.3 Gy and 87.3 and 123.4 Gy) Bland-Altman plots, 166Ho scout performed significantly better than 99mTc-MAA.Quantitative analysis showed that the overall mean differences in pre- minus post-treatment calculated absorbed dose for all lesions were 16.5 Gy for 99mTc-MAA and 0.2 Gy for 166Ho-scout .Quantitative analysis of the normal liver absorbed dose showed that the mean difference in the pre- and post-treatment calculated absorbed dose was 1.3 Gy for 166Ho-microspheres instead of 99mTc-MAA. The qualitative and quantitative analysis showed that the agreement between 166Ho-scout and 166Ho-therapeutic dose was significantly superior to 99mTc-MAA.This study showed that treatment planning prior to radioembolization can be improved by using a scout dose of The difference in agreement between scout dose distribution and treatment distribution is subject to several influencing factors. First, as stated before, catheter positioning is known to be a key factor . In orde166Ho-scout and 166Ho-therapeutic dose was better than with 99mTc-MAA. In addition, the mean of the overall agreement scores of all procedures of 166Ho-scout was significantly higher compared to 99mTc-MAA.Secondly, the assumption of a homogeneous distribution in the normal liver and lesion segmentations is not representative, leading to an overestimation of agreement. From a clinical perspective, normal liver-absorbed dose and tumor-absorbed dose are the most important parameters in terms of safety and efficacy. However, the VOIs of the normal liver compartment cover a much larger volume than the lesion VOI’s and therefore the level of overestimation of the agreement will be higher for this part of the analysis. In the qualitative analysis, agreement was visually analyzed on a more detailed, sub-segmental level. Interestingly, this analysis showed that in 65% of the 23 procedures, overall agreement between Thirdly, the width of the standard error of the 95% limits of agreement is strongly influenced by the number of analyzed procedures. Due to strict exclusion criteria, only 23 procedures were eligible for analysis. A larger study would require more patients to undergo two types of treatment-planning administrations, which is a burden to the patients, costly and arguably unethical.166Ho and 99mTc-MAA is inherently different in terms of background noise and resolution, which could have influenced both the qualitative and quantitative analyses. These inherent differences are unavoidable for conducting this study. In addition, the type of gamma camera, reconstruction software and matrix size was not uniform across the study. We think the influence of these factors on the outcome of the analyses is limited. A more important factor is the interval between injections. The injections of 166Ho-scout and 166Ho-therapeutic dose were performed on the same day. It is possible that the scout dose injection induced embolic effects that influenced the distribution of 166Ho-microspheres during the treatment injection later that day. The majority of the analyzed patients were treated with radioembolization for colorectal liver metastases that are known to be relatively hypovascular tumors. Especially in these patients, embolic effects may lead to alterations in microsphere distribution [166Ho-scout and 166Ho-therapeutic dose. Regardless of the type of scout-dose, embolic effects can be experienced during 166Ho-radioembolization, even though the number of 166Ho-microspheres injected for therapy (approximately 30 million) is lower than the typical number of resin 90Y-microspheres injected (estimated 50 million) [99mTc-MAA  and 166Ho-therapeutic dose may have led to interval changes in flow dynamics and thus altered distribution. Any type of scout dose performed on the same day as treatment may be more predictive than scout doses injected earlier. The difference in time interval may also result in 99mTc-MAA being administered by another operator than the operator injecting the 166Ho-therapeutic dose. Administration speed and technique may differ between operators. Also, the administration box used for 166Ho-scout and therapy administration with the possibility to intermittently change between contrast and microsphere injections was not available for 99mTc-MAA injections.Lastly, technical aspects may have influenced the outcome of this study. Imaging of ribution . This mamillion) . On the 166Ho-scout is that it is not as readily available as 99mTc-MAA, which can be easily extracted from a generator. 166Ho-scout needs to be ordered, activated in a nuclear reactor and delivered to the treatment site.A downside of 166Ho-scout. Elschot et al. showed that a scout dose of 166Ho-microspheres is more accurate for lung shunt calculation on SPECT/CT than 99mTc-MAA [166Ho-scout. They showed that extrahepatic depositions did not lead to any adverse events in their study. This supported the previously published findings by Prince et al. [This study focused on intrahepatic distribution, but lung shunt calculation is another important aspect of 9mTc-MAA . This ree et al. , 20.99mTc-MAA lacks dosimetric accuracy and needs improvement in order to increase the clinical benefit of radioembolization treatment. A recent study by Kafrouni et al. showed that body surface area (BSA) based activity planning  often leads to under-dosing, most likely because it insufficiently corrects for the tumor-to-normal (T/N) uptake ratios [99mTc-MAA is used to account for distribution differences between tumor and non-tumor compartments. This method is known to be a more personalized and accurate treatment planning method. However, the predictive value of 99mTc-MAA seems limited in many cases and the partition model is rarely used in clinical practice. The therapeutic activity choice in our treatment is based on one-compartment modeling or the so-called MIRD model, using an average absorbed dose to the target volume without differentiation between tumor and normal tissue absorbed doses. Analysis of 166Ho-scout and 166Ho-therapeutic dose imaging, together with treatment outcomes in terms of efficacy and safety, will lead to threshold values for personalized dosimetry-based treatment planning, which ultimately needs to be validated in prospective clinical studies. This study showed that 166Ho-scout may serve as a predictive “biomarker” for safe and effective treatment.Individualized treatment planning becomes increasingly important for patients who are selected for radioembolization. However, pre-treatment activity planning based on e ratios . Despitee ratios . As an a166Ho-scout agreed better with the distribution of the therapy dose than 99mTc-MAA, although the comparison has some inherent limitations. These results support the use of a scout dose of 166Ho-microspheres for radioembolization treatment planning.In conclusion, both the qualitative and quantitative analyses showed that the intrahepatic distribution of the


TDRKH-AS1 in CRC tissues and identified its potential targets. We found that TDRKH-AS1 is upregulated in majority of CRC patients, which is also significantly correlated with their malignant characteristics and their dismal prognoses. The high expression of TDRKH-AS1 can promote cancer cell proliferation substantially and invasion based on in vitro experiments. We also recognized that the TDRKH-AS1 targets the β-catenin in the Wnt signaling pathway to exert its carcinogenic activity. TDRKH-AS1 could serve as a promising prognostic predictor and a potential therapeutic target for further early diagnoses and treatments via a non-invasive method.Colorectal cancer (CRC) is a common cancer worldwide, with a lower 5-years survival rate. Recently, long non-coding RNAs (lncRNAs) have been well-studied as the oncogenes or the tumor suppressors in multiple malignancies, including CRC. However, their biological functions and potential mechanisms in human cancer remain unclear. Here, we evaluated the expression of  Colorectal carcinoma (CRC) is one of the most common malignancies with high mortality rates in the world, especially in the United States and European countries since last century , 2. AccoTDRKH-AS1 copy number and the survival rates of lung adenocarcinoma patients was reported in a high-throughput computational study  is a gene fragment that is more than 200 nucleotides and has no or weak possibility to be utilized for producing proteins . The expal study . TDRKH-ATDRKH-AS1 in CRC patients and carried out its biological function in the Wnt signaling pathway based on the patient data from “The Cancer Genome Atlas” (TCGA) database. TCGA is a government-funded project that provides publicly available data sets to help improve diagnostic methods, treatment standards, and ultimately cancer prevention from major cancer-causing genomes. The Wnt signaling pathway is a set of signal transduction pathways with multiple downstream channels stimulated by the binding of the ligand-protein Wnt and the membrane protein receptor. The extracellular signals are required to be transmitted into the cell through the activation of intracellular receptors on the cell surface ] at 37°C with a concentration of COThe cell counting kit-8 (CCK-8) assay  was used for evaluating the cell proliferation in the LoVo cells and HCT-116 cell lines by following the instructions. Approximately 1,000 cells per well were seeded into three wells on a 96-well plate, and 10 μl of CCK-8 was added to each well after being cultured for 24, 48, and 72 h. The cells were then analyzed for absorbance [optical density (OD) value] at 450 nm.siTDRKH-AS1 and the HCT-116 cells transfected with TDRKH-AS1 were added to the upper chamber containing RPMI1640 culture medium, and the lower chamber was filled with a cell suspension with 10% FBS. The LoVo cells transfected with negative control small interfering RNA (siRNA)  and MOCK , and the HCT-116 cells transfected with an empty vector  were used as the negative controls for each cell line. The LoVo cells transfected with an empty vector (pcDNA3.1) carrying siHOTAIR or siFEZF1-AS1 and the HCT-116 cells transfected with an empty vector (pcDNA3.1) carrying HOTAIR or FEZF1-AS1 were used as the positive controls for cell-line-based Transwell assay experiments  was used to extract total RNA from the cell lines. The complementary DNA (cDNA) library was obtained from the reverse transcription using the reverse transcriptase kit . The 10-μl system was placed in a 384-well plate for the quantitative real-time polymerase chain reaction inside the LifePro Thermal Cycler . The RNA level was monitored by 7900 Real-Time PCR System with the SDS v.2.3 software sequence detection system  using the SYBR Green  as reference. β-Actin served as the internal reference for t method .Cells were cotransfected with β-catenin luciferase reporter constructs based on the backbone of luciferase plasmid  and Renilla plasmid  according to the manual instruction. Cell lysis and luciferase activity were measured using the Luc-Pair™ Dual-Luminescence Assay Kit 2.0  according to the manufacturer's instructions.5 cells/ml and seeded on six-well plates (2 ml/ml). After cell adhesion, the cells were transfected with the TDRKH-AS1 interference sequence or the control sequence. After 48 h of culture, two groups of cells were collected and washed with PBS. Furthermore, cells were blocked using 2% FBS for 30 min and then washed with PBS. The cells were then dyed and placed in the dark for 30 min. The apoptosis rate of each group was determined by flow cytometry. Cells were treated and collected in a similar manner, and then, the cells were precooled with 75% ethanol and fixed in a refrigerator at −20°C overnight. The content of DNA was detected by propidium iodide (PI) staining, and the proportion of each phase was calculated by the software.Logarithmic CRC cells were adjusted to 3 × 10t-test and receiver operating characteristic curve analysis were used for analyzing the differences between the two groups. All the assays were repeated at least three times. Statistics were considered to be at a significant level when p < 0.05, where *p < 0.05, **p < 0.01, and ***p < 0.001.Statistical analysis was carried out using GraphPad Prism (version 8.2.1) software. Two-way TDRKH-AS1, the correlation coefficient (R) was calculated between TDRKH-AS1 and other genes in the CRC dataset from TCGA. Further, genes were ranked by their coefficients. The enrichment score (ES) for each pathway was calculated, and the significant level of the ES was also estimated from gene set enrichment analysis (GSEA) (To identify the relevant pathways that were regulated by s (GSEA) , 20. Thes (GSEA) .https://gdc.cancer.gov/) database. The individual demographic information and disease progress  were extracted and sorted from TCGA . Ge. Gehttps factors . The Empsoftware . Gene fuTDRKH-AS1 using the 71 CRC patients' RNA sequencing data from TCGA database. The fluctuation in the expression of the RNAs was focused on CRC between the patients in later stages with the initial stage , tumor size in centimeters , and depth of tumor in the first groups . We further identified that the TDRKH-AS1 was significantly upregulated in CRC tissues compared with the corresponding adjacent non-tumor tissue , siTDRKH-AS1 1 (siT-1) to siT-5, which were able to knock down the expression of the TDRKH-AS1 through silencing it. In the Transwell assay, the results showed that interfering with TDRKH-AS1 expression could inhibit cell invasion in the HCT-116 cell line, and siT-1 and siT-3 had a better knockdown effect  in CRC from GSEA  assay indicated that TDRKH-AS1 influenced the Wnt signaling pathway at the RNA level . Af. AfTDRKHNA level . The oncce versa . Further-catenin .MIR22HG  data from TCGA focused on exon sequences   , 31, Fri (DKK-1) –34, Wnt  (DKK-1) , 36, Ceb (DKK-1) . These pThe datasets generated for this study can be found in The Cancer Genome Atlas (TCGA) in CRC subgroup. This data can be found here: Carlson . org.Hs.The studies involving human participants were reviewed and approved by The Affiliated Hospital of Jiangnan University. The patients/participants provided their written informed consent to participate in this study. TCGA Ethics and Policies was originally published by the National Cancer Institute.JwZ and JyZ conceived and performed the experiments and wrote the manuscript. MS collected the patient samples and interpreted the data. YaJ, JlZ, YeJ, YY, and LZ analyzed the experimental data. JyZ performed the RNA-seq data analyses and visulizations. All authors read and approved the final draft of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Research on the brain-gut-microbiota axis has led to accumulating interest in gut microbiota dysbiosis and intestinal barrier dysfunction in Alzheimer’s disease (AD). Our previous studies have demonstrated neurotoxic effects of 27-hydroxycholesterol (27-OHC) in in vitro and in vivo models. Here, alterations in the gut microbiota and intestinal barrier functions were investigated as the possible causes of cognitive deficits induced by 27-OHC treatment.d-lactate. Systemic and intestinal inflammation were evaluated by IL-1β, TNF-α, IL-10, and IL-17 concentrations as determined by ELISA. The fecal microbiome and short-chain fatty acids (SCFAs) were analyzed using 16S rDNA sequencing and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Tight junction proteins were evaluated in the ileum and colon by qRT-PCR and Western blots. Tight junction ultrastructure was examined by transmission electron microscopy.Male APP/PS1 transgenic and C57BL/6J mice were treated for 3 weeks with 27-OHC  and either a 27-OHC synthetase inhibitor  or saline. The Morris water maze and passive avoidance test were used to assess cognitive impairment. Injuries of the intestine were evaluated by histopathological examination. Intestinal barrier function was assessed by plasma diamine oxidase (DAO) activity and Roseburia and reduced fecal levels of several SCFAs in 27-OHC-treated mice. Meanwhile, co-treatment with ANS reduced intestinal inflammation and partially preserved intestinal barrier integrity in the presence of 27-OHC.Treatment with 27-OHC resulted in severe pathologies in the ileum and colon. There was impaired intestinal barrier integrity as indicated by dilated tight junctions and downregulation of tight junction proteins, including occludin, claudin 1, claudin 5, and ZO-1, and signs of inflammation . Fecal 16S rDNA sequencing and taxonomic analysis further revealed a decreased abundance of The current study demonstrates for the first time that 27-OHC treatment aggravates AD-associated pathophysiological alterations, specifically gut microbiota dysbiosis and intestinal barrier dysfunction, which suggests that the gut microbiome and intestinal barrier function warrant further investigation as potential targets to mitigate the neurotoxic impact of 27-OHC on cognitive function and the development of AD. Alzheimer’s disease (AD) is a highly prevalent neurodegenerative disease in older people. It is clinically characterized by progressive cognitive impairment and irreversible impairment of the ability to perform activities of daily living . It is pAlterations in gut microbiota have been reported to be intimately involved in the pathogenesis of AD. Human studies have reported that the distinct gut microbiota of AD patients is characterized by enriched in Enterobacteriaceae as well as abundant Bacteroides, Actinobacteria, and Selenomonadales as compared with predementia stage mild cognitive impairment (MCI) or healthy controls . MoreoveThe bidirectional networks between the brain and gut microbiota are maintained through metabolic, immune, endocrine, and neuronal pathways . Recent On the one hand, the intestinal barrier permeability may be altered by endogenous or exogenous factors as an outcome of inflammatory processes, which may cause the introduction of undesired pathogens into the body; on the other hand, neuroinflammatory conditions are associated with blood-brain barrier (BBB) disruption, which may fail to maintain the permeability and ensure homeostasis of the central nervous system (CNS) . ConsequDespite being relatively stable, the gut microbiota composition gradually undergoes changes due to lifestyle, geographical location, and dietary habits . An incr27-OHC has been suggested to be involved in the pathogenesis of gastrointestinal and neurodegenerative diseases  due to iOur recent study  demonstrAnastrozole (ANS), an anti-breast cancer pharmaceutical, was tested previously in mice in vivo by Mast et al.  and was However, as reported by the aforementioned articles, some findings of the in vivo studies could not be replicated in differentiated CaCo-2 cells, human neuroblastoma cells, or rat glioma cells. Therefore, the underlying mechanisms by which 27-OHC exerts its neurotoxic effects are still unclear, and whether 27-OHC can trigger cognitive deficits through inflammatory responses in the gut–brain axis has rarely been examined in vivo. Bearing this in mind, we considered it essential to extend our research from cell models to animal models.The mutations in presenilin 1 and amyloid precursor protein (APP) genes are the most commonly recognized triggers of familial AD, which led to the development of the APP/PS1 transgenic mouse model that mimics some of the pathology in AD and presents with increased Aβ1-42 production . In thisSpecific pathogen-free (SPF) male APP/PS1 transgenic and sex- and age-matched wild-type C57BL/6J mice, 6 months old, weighing 30–35 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.  2017-0022). The mice were housed in standard individual ventilated cages in a temperature- (22–25 °C) and humidity- (50–60%) controlled room with a 12 h dark-light cycle. Food and water were available ad libitum.Our previous study has already investigated the effects of intravenous injection of 27-OHC in rats . Howevern = 10/group) were randomly assigned to seven groups . After treatment for 3 weeks, behavioral tests were carried out.The 27-OHC doses used for the current study were based on the known effective dose in rats (70 μM/day) , the phyn = 10/group) and matched wild-type C57BL/6J mice  were randomly divided into five groups: group I: WT control (C57BL/6J mice); group II: APP/PS1 control; group III: WT mice treated with 27-OHC (selected effective dose for subcutaneous injection); group IV: APP/PS1 mice treated with 27-OHC (selected effective dose for subcutaneous injection); group V: APP/PS1 mice treated with anastrozole ; group VI: WT mice cotreated with 27-OHC (selected effective dose for subcutaneous injection) plus ANS ; and group VII: APP/PS1 mice cotreated with 27-OHC (selected effective dose for subcutaneous injection) plus ANS . The APP/PS1-control and C57BL/6J-control groups received the same volume of 0.9% normal saline alone according to the same schedule.Next, the APP/PS1 mice  divided into four quadrants. A hidden escape platform (9 cm in diameter) was placed 2.0 cm below the water in the center of one quadrant. The water was colored with titanium dioxide for the C57BL/6J and APP/PS1 mice and adjusted to 22 ± 1 °C. The tank was placed in a dimly lit and soundproof room with various visual cues.The orientation navigation test was performed for five consecutive days, and the mice were released into the water from different starting positions and allowed to swim freely until they reached the hidden platform. This was repeated four times each day with an interval of 30 min. The latency to reach the platform and swimming trajectory were recorded by a computer-controlled video tracking system  for a maximum duration of 90 s. Mice that failed to reach the hidden platform within 90 s were placed on it for 15 s. The escape latency was determined as the time from the starting position to the platform. Afterwards, the spatial probe test was carried out. The platform was removed on the sixth day. The mice were released from a position opposite from where the platform used to be and allowed to swim for 90 s in the maze. The time spent in the target quadrant, the time spent crossing where the platform was originally located, swimming speed, and swimming trajectory were recorded.The passive avoidance test is a fear-motivated test used to evaluate learning and retention memory. It is conducted in an apparatus that consists of two identical compartments (light and dark) 25 × 25 × 25 cm in size with a sliding door and grid-metal floor. The test enables mice to learn to restrain their innate behavior through moving from the illuminated compartment to the dark compartment. During the passive avoidance preacquisition trial, the mice were placed into the light compartment for a 5-min adaptation. The sliding door was automatically closed as soon as the mouse completely entered the dark compartment. Subsequently, the mouse was subjected to an electrical foot-shock (0.5 mA for 3 s) via the floor grid. The latency to enter the dark compartment was detected and analyzed and mice with latencies longer than 300 s were omitted from the study.The retention memory test trial was tested 48 h after the acquired response had been established by recording their latency to enter the dark compartment. The latency to enter the dark compartment and frequency of entries into the dark compartment were recorded and directly linked to the retention memory. A greater latency and lower frequency of entry indicates better memory.ethylenediaminetetraacetic acid (EDTA)-plasma samples were collected and stored at − 80 °C until use. Following blood collection, the mice were perfused transcardially with ice-cold 0.2 M phosphate buffer solution (PBS). Their brains and the entire intestine were removed and fixed in 4% paraformaldehyde at 4 °C followed by processing for paraffin embedding and sectioning for histopathological analysis. Meanwhile, at necropsy, tissue samples from the brain, liver, kidneys, spleen, and intestine that showed gross changes were examined. The organ coefficient is expressed as the ratio to body weight to account for differences in organ weight-related effects and overt signs of toxicity.APP/PS1 and C57BL/6J mice were anesthetized with 5% chloral hydrate . Fasting blood was drawn via cardiac puncture with a syringe. After being centrifuged at 3000 g for 15 min at 4 °C, serum and n = 6/group). The Aβ load in the brains was assessed using Bielschowsky silver stain (BSS), which was performed according to the previously published protocols [electron microscope .A part of the fixed brain as well as the distal ileum and proximal colon tissues were embedded in paraffin, cut into 5 μm sections, stained with hematoxylin and eosin (HE), and examined by light microscopy (rotocols . A portin = 6/group) according to our previously reported protocol [High-performance liquid chromatography-mass spectrometry  was used to measure the plasma and brain levels of 27-OHC (protocol .n = 6/group). Each experimental sample was run in triplicate. The data were recorded at 450 nm using a microplate reader .The levels of Aβ1-40 and Aβ1-42 in the plasma and brains were determined by ELISA kits  in accordance with the manufacturer’s instructions (n = 6/group).Plasma and liver levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured by commercial kits . All of the procedures were performed according to the manufacturers’ protocols (n = 6/group).Levels of IL-1β, IL-10, TNF-α, and IL-17 in the plasma were measured by enzyme-linked immunosorbent assay ELISA kits  following the manufacturers' instructions  and tight junction proteins  as well as intestinal inflammatory cytokines (IL-1β and IL-10) were measured to assess intestinal barrier dysfunction.d-lactate in serum were measured by commercial kits . All of the procedures were performed according to the manufacturers’ protocols (n = 6/group).DAO activity and n = 6/group) and Western blots (n = 6/group). RNA extraction was performed according to the manufacturer’s instructions using the SV Total RNA Isolation System . The RNA concentration was measured and 1 μg of RNA was reverse-transcribed using a RevertAid First Strand cDNA Synthesis kit  according to the manufacturer’s instructions. For relative quantification of mRNA levels, qRT-PCR was performed on a Real-time PCR Detection System . All samples were measured in triplicate. The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the reference for standardization. Quantitative measurements of target gene levels relative to controls were performed with the 2-ΔΔCt method.The mRNA and protein expression of occludin, claudin 1 (CLOD-1), claudin 5 (CLOD-5), and ZO-1 in the ileum and colon were measured by quantitative real-time polymerase chain reaction . The concentrations of the protein extracts were determined by a BCA assay kit . Proteins (50 μg per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel by electrophoresis (SDS-PAGE) and transferred onto PVDF membranes . The membranes were blocked with 5% bovine serum albumin prepared in TBS  for 1 h at room temperature, then incubated with the primary antibodies overnight at 4 °C, followed by the HRP-conjugated secondary antibodies . All primary antibodies were as follows: anti-ZO-1 , anti-Occludin , anti-Cldn-1 , anti-Cldn-5 , and β-actin . Protein bands were visualized by an enhanced chemiluminescence kit . The density of each band was measured using Image System Fusion FX  and normalized to β-actin levels. The experiments were repeated three times. The plots for Western blot were individual samples representative for each group.n = 6/group).Levels of IL-1β, IL-10, TNF-α, and IL-17 in the distal ileum specimens and proximal colon were also measured by ELISA kits  following the manufacturers’ instructions (n = 6/group) and subjected to DNA isolation using a Bacterial DNA Kit . The DNA concentration was determined using a Nanodrop 1000 . The 16S rDNA gene library preparation was conducted by using PCR amplification of the V3–V4 region. All libraries were sequenced using an Illumina HiSeq2500 platform  at Biomarker Technologies, Beijing, China. Bioinformatic analyses were performed to compare the differences in the gut microbiota between the indicated experimental groups.At the end of the treatment, fecal samples were collected (n = 6/group).Fecal samples (50 mg) were mixed with an aliquot of 1000 μL of methanol/water (v/v = 1:1) mixture and then vortexed for 1 h and centrifuged at 132,000 r/min for 10 min. Then, 50 μL supernatant was transferred into a 1.5 mL centrifuge tube and mixed with 50 μL of propionic acid-d2 (1 μg/mL), which acts as an internal standard. Samples were then derivatized with 3NPH and centrifuged at 132,000 r/min for 10 min, and the supernatant was collected. An aliquot (5 μL) of the resulting derivatized material was injected into an ultra-performance liquid chromatograph (Waters UPLC I-Class) coupled to a tandem quadrupole mass spectrometer detector (Waters XEVO TQ-S Micro). Standard stock solutions of acetate, propionate, isobutyrate, butyrate, valerate, isovalerate, caproate, and heptanoic acid (ca. 1 mg/mL) were prepared by dissolving specific amounts of authentic standards in chloroform-methanol . The working standard solutions of the short-chain fatty acids (SCFAs) were serially diluted with chloroform-methanol  to obtain the concentrations needed for calibration curve standards . The estimate of the relative concentrations was based on the ratio of the area under the curve of the internal standard for each SCFA  test. If the data followed a normal distribution, data are presented as the mean ± standard deviation and parametric tests (one-way ANOVA for three or more groups) were performed; for ANOVA, least significance difference (LSD) testing was carried out for post hoc analysis. If the data were not normally distributed, the data are presented as the median (interquartile range) and nonparametric tests  were used by applying SPSS 19.0 software  and Prism 6.0 . Repeated measures analysis of variance (ANOVA) were performed for the water maze data as appropriate. Correlations were analyzed by Spearman’s test. For 16S rDNA sequencing, sequences with ≥ 97% similarity were assigned to the same operational taxonomic units (OTUs). Representative sequences for each OTU were screened for further annotation. For each representative sequence, the Silva Database was used based on the mothur algorithm to annotate the taxonomic information. The following downstream data analyses were conducted in R software. Alpha diversity was applied for analyzing the complexity of species diversity for a sample through three indices, including Chao1, Shannon, and Ace. ANOVA was performed to evaluate α-diversity among the different groups. Beta diversity analysis was used to evaluate differences of the samples in species complexity. The linear discriminant analysis (LDA) effect size (LEfSe) method was performed to detect differentially abundant features with statistical significance and biological relevance among different groups and an LDA score of > 4 was used as the threshold. Metastats analysis was used to compare bacterial relative abundance between different groups.P < 0.05, Fig. P < 0.05, Fig. P < 0.05, Fig. P < 0.05, Fig. P < 0.05, Fig. The body weight of the treated C57BL/6J mice tended to decrease throughout the experimental period, albeit without significant differences Fig. a. MoreovP < 0.05, Fig. P < 0.05, Fig. P < 0.05, Fig. P < 0.05, Fig. P < 0.05, Fig. The Morris water maze and passive avoidance test were used to investigate whether subcutaneous injection of 27-OHC causes learning and memory deficits in mice. Regarding the Morris water maze, mice treated with doses of 27-OHC higher than 1.65 mg/kg displayed significant longer escape latency to the target platform .As illustrated in Fig. P < 0.05). In addition, 27-OHC treatment could cause significant but different changes of plasma and liver cholesterol levels . Interestingly, the ANS-treated APP/PS1 group preferentially performed a selective search for the platform and had a shorter distance to the platform than the 27-OHC-treated APP/PS1 group .We found that 27-OHC administration to APP/PS1 and WT mice significantly increased their latency to locate the platform , while the ANS-treated APP/PS1 group completely reversed this trend. 27-OHC + ANS-treated mice showed no significant differences in the number of target crossings or the target-quadrant abidance in comparison to the control mice (P > 0.05). For swimming speed, the ANS-treated APP/PS1 group had a significantly increased speed relative to both the control groups and the 27-OHC-treated groups .In the spatial probe test, compared to the WT mice, the APP/PS1 group mice swam more randomly throughout the tank, suggesting poor memory retention of the location of the platform. The 27-OHC-treated APP/PS1 or WT group showed an obvious decrease in the number of target crossings Fig. d and tarP< 0.05), suggesting that 27-OHC treatment could induce learning and memory impairment. Both the ANS alone-treated and 27-OHC + ANS-treated groups had significantly prolonged latencies of entry into the dark area and had decreased frequencies of entries into the dark compartment (P < 0.05). These results indicated that the high dose of 27-OHC could result in spatial learning and memory capability impairment while inhibition of 27-OHC synthesis could prevent or reverse the cognitive deficits induced by 27-OHC in the APP/PS1 double transgenic mice.In the passive avoidance test, compared to the APP/PS1 and WT control groups, the latency to enter the dark area was significantly shortened and the frequency of entries into the dark compartment was increased in the 27-OHC-treated group  were displayed at a 97% similarity level and shared among all samples Fig. a. The ovP < 0.05). To assess the overall structure of the gut microbiota, β diversity analysis for interindividual microbiota relatedness using unweighted UniFrac distance demonstrated that the structure and composition of the microbiota differed significantly among groups  effect size (LEfSe). We used a logarithmic LDA score cutoff of 4.0 to identify important taxonomic differences among the five groups and the representative cladogram showed variant taxa at different taxonomic levels Fig. a, b. Herice Fig. c, d. As P < 0.05). However, the isobutyrate and isovalerate levels were significantly higher in the 27-OHC-treated mice than in the control and ANS-treated mice (P < 0.05). There were no significant differences in heptanoic acid among the groups (P > 0.05).We also explored alterations of fecal SCFA profiles caused by 27-OHC Fig. a–h. Our To investigate the impact of 27-OHC on the intestinal pathology, histopathological changes of the ileum and colon were examined. As shown in Fig. Next, we applied transmission electron microscopy for ultrastructural analysis of tight junctions and microvilli in the ileum and colon. Figure d-lactate were measured as indicators of intestinal permeability. As shown in Fig. d-lactate were both significantly increased in the 27-OHC-treated APP/PS1 group . The ANS and 27-OHC + ANS groups had significantly lower levels of serum DAO and d-lactate than those of the control groups .To evaluate the severity of intestinal barrier dysfunction, serum DAO and P < 0.05), except that no significant changes were observed in the protein expression levels of occludin (P > 0.05). In contrast, the ileal and colonic expression was markedly increased as a result of ANS or 27-OHC plus ANS treatment (P < 0.05).The ileal and colonic gene and protein expression of tight junction proteins was further analyzed to confirm the detrimental effects of 27-OHC treatment on the intestinal integrity. As shown in Fig. P < 0.05) compared with the WT and APP/PS1 control groups. The ANS- and 27-OHC + ANS-treated APP/PS1 groups had significantly lower levels of IL-1β than the 27-OHC-treated APP/PS1 group (P < 0.05). Moreover, the APP/PS1 mice also had significantly higher IL-1β levels in the plasma, ileum, and colon than the WT mice. In regard to TNF-α, similar patterns of differences were also observed in the plasma, ileum, and colon levels of TNF-α among the different groups except that 27-OHC treatment did not induce a significant increase in WT mice. However, no significant changes were observed in the IL-10 levels among the different groups , but ANS treatment could induce significantly increased IL-17 levels in the plasma, ileum, and colon .We evaluated systemic and intestinal inflammation by measuring the levels of the pro-inflammatory cytokines IL-1β and TNF-α as well as the levels of the anti-inflammatory cytokines IL-10 and IL-17 in the plasma and intestinal tissue. As shown in Fig. Hypercholesterolemia in midlife is considered to be a modifiable risk factor for the development of AD and dementia . In addiAs the most abundant oxysterol and BBB-permeable cholesterol metabolite in the blood, 27-OHC has been reported to induce cognitive impairment. Research from our laboratory suggested that 27-OHC induces neurotoxic effects in the brain in vivo as well as in neuronal cells in vitro, involving mechanisms of neuroinflammation , pyroptoThe main findings of this study were as follows: (i) 27-OHC induced gut and circulating inflammation and alterations in the gut microbiome and SCFAs, disrupting the intestinal barrier, which may allow the passage of bacterial products and subsequently aggravate the brain amyloid plaque burden and cause cognitive deficits. (ii) Cotreatment with an inhibitor of 27-OHC synthesis, ANS, reduced gut and systemic inflammation and preserved the structure of the intestinal barrier. In conclusion, we have provided new experimental evidence for a critical role of 27-OHC in gut dysbiosis and impairment of intestinal barrier permeability that aggravates inflammation and leads to cognitive deficits and an increased Aβ load in APP/PS1 mice. These results also suggest that pharmacological treatment with ANS may preserve the integrity of the intestinal barrier, alleviate inflammation, and reverse the cognitive deficits induced by 27-OHC.Cognitive deficits are the main manifestation of AD . In the A disruption in the gut microbial composition has been reported to be associated with many neurological disorders including AD . SeveralShen et al.  found thRoseburia abundance at the genus level and the fecal levels of some SCFAs were significantly lower in the 27-OHC-treated mice than the control and ANS-treated mice. Roseburia is one of the most abundant butyrate-producing bacteria and accounts for 0.9 to 5.0% of the total microbial load [Roseburia has been reported to be decreased in many intestinal disorders [Roseburia, fecal levels of propionate, butyrate, caproate, valerate, and acetate were also significantly decreased in 27-OHC-treated mice. Roseburia has also been reported to exert anti-inflammatory effects in in vivo and in vitro models by inducing anti-inflammatory cytokine production and decreasing the production of proinflammatory cytokines [Further inspection showed that ial load . The abuisorders  as well isorders , indicatisorders . Meanwhiisorders , which aytokines .Faecalibacterium, Ruminococcaceae, and Roseburia abundance [Meanwhile, significant increases in isobutyrate and isovalerate in 27-OHC-treated WT and APP/PS1 mice were also observed. In a randomized crossover study, the fecal concentrations of both isobutyrate and isovalerate were significantly decreased in subjects with metabolic syndrome, including dyslipidemia, insulin sensitivity, and abdominal obesity, in response to dietary fiber intervention , indicatbundance . TherefoThere is growing evidence indicating that cholesterol auto-oxidation products, namely oxysterols, can promote and stimulate intestinal inflammatory diseases with pro-inflammatory properties and by inducing epithelial barrier disturbance. A recent study showed the ability of a combination of dietary auto-oxidation oxysterols to induce the loss of intestinal epithelial layer integrity in monolayers of differentiated CaCo-2 cells , with sud-lactate and DAO; all of which indicated that the intestinal barrier dysfunction became severe after treatment with 27-OHC. Disruption of the intestinal barrier will undoubtedly lead to increased intestinal permeability and systemic exposure to bacterial products such as lipopolysaccharide (LPS) and other luminal endotoxins, pathogens, and antigens, which could trigger systemic inflammatory responses [For example, treatment with 7-ketocholesterol, an oxygenated cholesterol product present in foodstuffs, reduced epithelial barrier function and diminished IL-10 mRNA expression, an anti-inflammatory cytokine, suggesting oxysterols may contribute to the dysregulation of epithelial barrier function and an inappropriate inflammatory response . Here, wesponses .Accumulated evidence has reported that impaired intestinal barrier integrity is mechanistically involved in the pathological progression of neuroinflammation and cognitive deficits . Gut-bloThe antagonistic actions of ANS against 27-OHC treatment have been described in our previous studies , 22. In In summary, 27-OHC treatment induced dysfunction of the intestinal barrier, characterized by an increased intestinal inflammatory response, loss of tight junction proteins, and gut microbiota and SCFAs dysbiosis, which resulted in elevated systematic inflammation, brain Aβ deposits, and cognitive deficits. Cotreatment with ANS ameliorated the systematic and local inflammatory responses, partially preventing the damage to the tight junction proteins and brain induced by 27-OHC. The deterioration of the intestinal barrier and gut microbiota changes induced by 27-OHC may be some of the mechanisms leading to cognitive deficits. This provides new insight into exploring the neurotoxic effects of 27-OHC and suggests that ANS-based pharmacotherapies, such as a coadjuvant strategy, could be potentially useful to treat brain disorders characterized by 27-OHC overload.


There is a need of comprehensive standardized diagnostic assessment tools of psychopathology that match recent changes in diagnostic classification systems, such as the 5th edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5). Therefore, the computer-assisted DIA-X-5 was developed and its test-retest reliability was explored. The DIA-X-5 is based on the DIA-X/M-CIDI  which referred to the 4th edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV).N = 60, age: 15–67) was interviewed twice with the computer-assisted DIA-X-5 interview, on average nine days apart, by trained and blinded interviewers. The DIA-X-5 is a standardized instrument for research purposes covering symptoms, syndromes and diagnoses from eleven classes of mental disorders according to the DSM-5 with matching F codes of the 10th edition of the International Classification of Diseases (ICD-10).A convenience sample (Kappa values ranged from 0.90 for post-traumatic stress disorder to 0.30 for social anxiety disorder. For age of onset and age of recency, test-retest reliability as measured by intra-class correlation was satisfying with values above 0.90 for most disorders.Test-retest reliability of the DIA-X-5 syndromes and diagnoses were comparable to those of previous DSM-IV/DIA-X diagnoses for most disorders. Due to low case numbers for some diagnoses, further research in larger samples is required. The development of structured and standardized diagnostic interviews has considerably improved the reliability and validity of the assessment of mental disorders even when conducted by non-clinical (lay) interviewers , 2. WithWith the publication of the fifth revision of the DSM , diagnosIn response to the aforementioned changes, it has become necessary to provide a revised version of the DIA-X interview incorporating these changes, while maintaining the comparability to previous versions of the instrument and the implicit diagnostic algorithms to provide methodological consistency with previous studies and allow investigations into the effects of criteria changes on prevalence, onset, course and comorbidity findings. This paper presents the modification and extension process as well as test-retest reliability data of the new DIA-X-5.This section is structured into two major components. Firstly, the development of the DIA-X-5 interview is described, including its diagnostic coverage as well as its outer format. Secondly, the reliability is examined in a retest study for which results are presented in the current manuscript.The DIA-X/M-CIDI is a well-established standardized clinical interview  which waThe DIA-X-5 assesses symptoms, syndromes and diagnoses of eleven major classes of mental disorders for the lifetime and the past 12 month time frame, except for adjustment disorders and premenstrual dysphoric syndrome which are only assessed for the past 12 months. Diagnostic algorithms were reprogrammed to match recent changes in DSM-5  algorithFor reasons of consistency the content, format, rules and style of the past DIA-X/M-CIDI were maintained. Previous data have shown that even minimal changes might influence the response behavior of the subjects . ResponsThe structure of some disorder sections was modified to improve the interview flow given the changes in diagnostic criteria. First, in the substance use disorder sections , the separate assessment of abuse and dependence was omitted in favor of one comprehensive list covering all substance use disorder symptoms . Second, given the considerable changes surrounding somatoform disorders, this section has been changed to include questions assessing symptoms of somatic symptom disorder and omitting the previous probe questions assessing whether each individual symptom is “medically unexplained”.In the section of anxiety disorders, for panic symptoms and panic disorder the order of questions was changed, given the prominent introduction of a “panic attack specifier” in DSM-5 , which cIn disorder sections where variation in disorder symptomatology and severity are expected (e.g. mood episodes), the DIA-X-5 lifetime questions first ask for the presence of lifetime symptoms followed by questions regarding the worst lifetime episode; and finally, which of these symptoms have also been present in the past 12 months. In this way, past 12 month diagnoses assure that the full diagnostic criteria are met during the last year. Further, this additional probing allows for the derivation of partial remissions and severity coding for past year diagnoses.More extended changes in the DIA-X-5 include the following:A convenience sample of adolescents and adults (targeting age 14–65 years) was recruited by advertisement and in collaboration with clinical institutions. Participants were invited to participate in two diagnostic face-to-face interview sessions approximately one week apart. Upon arrival at the interview location, participants were informed about the procedures of the study and asked whether they would provide their written informed consent; for participants younger than 18 years of age, written informed assent/consent was obtained from both the adolescent and all legal guardians . Both interviews were conducted at the research institution  or at the university hospital by two independent interviewers, i.e. the second interview was conducted blinded to the results of the first interview. Individuals were instructed to answer the questions in the second interview disregarding their answers during the first interview.Interviews were conducted by trained clinical interviewers . The 25 interviewers had a mean age of 26 years and 23 were women. Each interviewer had sufficient experience in the computerized version of the DIA-X-5 having had received a standardized multiday interviewer training and underwent a practice and certification process. Participants received payment (30€) for their participation in both interviews.Diagnostic concordance for each disorder, i.e. agreement of diagnostic results of the test (T1) and the retest interview (T2), was calculated as the relative frequency of individuals with equal disorder classification on both assessment times. The agreement for disorder categories were only calculated if the diagnostic base rate was ≥5 . Diagnostic agreement was also computed for all core DIA-X-5 (stem items) symptom questions. For diagnoses with multiple stem items, at least one of the multiple stem items had to be affirmed to compute agreement between both interviews. Calculated coefficients were Jaccard index (JI ), a reveThe Jaccard Index (JI ) is the For this study, a reversed Jaccard Index (RJI) was developed. The RJI is the number of individuals with a negative disorder diagnosis on both DIA-X-5 assessments divided by the number of individuals with negative disorder diagnosis on at least one assessment. The RJI is an estimated lower bound for the probability that a non-case at one interview will be evaluated to be a non-case at another interview. A higher RJI indicates a higher probability of re-identification of a non-case at another time point. RJI was calculated in the following mannerJI and RJI are easy-to-interpret and symmetric indices which mean that no pre-defined order of assessments is assumed. Thus, non-cases of the first interview (T1) that became cases at the second interview (T2) are counted like non-cases of T2 that were cases at T1, both are equally hampering the indices JI and RJI.Cohen’s kappa  is the mIn the present study, for most disorders more cases emerged at T1 than at T2. Thus, also the bias-adjusted kappa  was comAssociations between time-related measures  were calculated as intraclass correlation coefficient (ICC). We used the one-way random-effects ICC  for absoAll analyses were conducted using Stata 14.2  and confM = 26.6 years) and included 44 women (73.3%). Most participants were attending university (58.3%) and were not married (93.3%). The mean time interval between both interviews was nine days, with a range of one to 36 days. For 46.7% of participants, the time interval was 7–9 days, for 81.7% of participants, the time interval was 4–13 days. For more details see Table The sample consisted of 60 participants, aged 15 to 67 years M = 6.6 yearsM = 32.9 min) and for depressive disorders (M = 16.1 min). Other sections that enquire only single or less prevalent disorders were considerably shorter, such as oppositional-defiant disorder (M = 1.8 min) and intermittend explosive disorder (M = 2.0 min).The average time to complete the DIA-X-5 excluding the sociodemographic section was 125.9 min (range 30.8–405.0 min). The duration of all sections of the interview is listed in Table BAK values were comparable to kappa values (deviation ≤0.03) and are thus not reported in the following sections.Test-retest reliability of DSM-5 diagnoses and diagnostic classes is displayed in Table For most disorders the JI ranged between 0.65 and 0.85. The highest JI, 1.00, was found for anorexia nervosa, second highest JI (0.89) was found for ‘any’ DSM-5 disorder. PTSD and tobacco use disorder had a JI of 0.83. Social anxiety disorder displayed the lowest JI with 0.25, followed by any obsessive compulsive disorder with 0.37.RJIs for most disorders were 0.80 and above. Anorexia nervosa displayed highest RJI with 1.00, followed by PTSD and cannabis use disorder with 0.98. The category of any anxiety disorder (without panic attack) revealed lowest RJI with 0.69, followed by ‘any’ DSM-5 disorder and any anxiety disorder  with an RJI of 0.76 each.Highest agreement with 100.0% was found anorexia nervosa, followed by a 98.3% agreement rate for PTSD and cannabis use disorder. Any DSM-5 disorder had an agreement of 91.7%. The categories of any anxiety disorder (without panic attack) and social anxiety disorder revealed the lowest agreement with 78.3 and 80.0% respectively. However, most diagnostic categories displayed an agreement of 90% and above. Most Cohen’s kappa values ranged between 0.70–0.85, with anorexia nervosa displaying the highest kappa (1.00), followed by PTSD (0.90), tobacco use disorder (0.89) and cannabis use disorder (0.88). The diagnosis of ‘any’ DSM-5 disorder had a kappa of 0.81. Cohens Kappa was lowest for social anxiety disorder (0.29), followed by any obsessive-compulsive disorder (0.51) and any anxiety disorder without panic attack (0.55). For discordant cases it was also tested whether for those disorders with all fulfilled criteria in one interview, at least the respective stem question was endorsed in the other interview. Of the two single disorders with low kappa, the respective stem question was always endorsed for social anxiety disorder. However, for obsessive-compulsive disorder (OCD) two out of three cases did not endorse the stem question in one interview although they fulfilled all criteria for a diagnosis in the other interview.JI = 0.94) and major depressive disorder (JI = 0.91) displayed the highest JIs. For most disorders, JIs ranged between 0.65 and 0.85. RJI was the highest for any drug use disorder - illicit drug consume (0.98), followed by tobacco use disorder – symptom item (0.95) and conduct disorder (0.94). Medication use – consume item had the lowest RJI with 0.64. For most disorders the RJI was 0.85 and above.Diagnostic agreement for the core DIA-X-5 stem items are shown in Table Agreement was highest for any drug use disorder – illicit drug consume (98.3%) and tobacco use disorder – symptom item (96.7%). Medication use – consume item had the lowest agreement rate of 73.3%. For most disorders, agreement was 80% and above.Kappa values were highest for any drug use disorder – illicit drug consume (0.96) and were also high for tobacco use disorder – symptom item (0.92). Separation anxiety disorder had the lowest kappa value with 0.43, followed by medication use – consume item 0.44. Stem items of most disorders had kappa values between 0.70 and 0.85.ICCs for age of onset, age of recency, and persistence are displayed in Table For most diagnoses, ICCs for age of onset were 0.90 and above. ICC for age of onset was highest for PTSD, panic disorder, single major depressive disorder and antisocial personality disorder with 0.99. ICC for onset was lowest for specific phobia with − 0.74  and also low for major depressive disorder with recurrent episodes (0.40).ICCs of age of recency were above 0.90 for most disorders. An ICC of 1.00 was found for recency of obsessive-compulsive disorder (behavior). Also, ICCs were high with 0.99 for tobacco use disorder, anorexia nervosa, binge eating disorder, panic disorder, specific phobia , single major depressive episode, possible psychotic disorder and ADHD (inattentive presentation). The lowest ICC for recency was shown by specific phobia  with 0.61.Most disorders revealed ICCs for persistence between 0.55 and 0.85. Persistence reliability was highest for specific phobia  with 0.96, followed by specific phobia  with 0.94. Lowest ICC for persistence was revealed by somatic symptom disorder with 0.16, followed by possible psychotic disorder with 0.20.The standardized assessment of symptoms, syndromes and diagnoses of mental disorders is essential for estimating the prevalence, onset, and course of mental disorders and determining their risk factors in epidemiologic research. Case definition in clinical and experimental studies also relies on reliable diagnoses. The standardized and fully computerized DIA-X-5 reveals good test-retest reliability for most DSM-5 diagnoses, stem items and time-related information in adolescents and adults.Although most of the DIA-X-5 diagnoses showed good to very good test-retest reliability, some diagnoses showed relatively low reliability. For these diagnoses we examined in more detail the response patterns on the level of diagnostic criteria and individual questions.The summary category of any anxiety disorder (without panic attack) reveals relatively low reliability because of low reliability indices for few specific anxiety diagnoses. For panic disorder, comparing each separate diagnostic criterion, there was no specific response pattern which changed from the first to the second interview. However, a change in the order of questions in this section may have affected subjects’ overall response behavior. In the DIA-X/M-CIDI, the panic attack stem question was followed by panic disorder questions only after which the panic attack symptoms were assessed. This order changed in the DIA-X-5, probing the panic attack symptoms before the panic disorder criteria, due to the prominent role of the panic attack specifier in DSM-5 . ReliabiFor social anxiety disorder, participants’ responses varied particularly for avoidance (5 of 12 discordant cases) and duration of anxiety (6 out of 12 discordant cases). Social anxiety disorder consists of a long criterion list and 9 out of 12 discordant cases were discordant only in one criterion. Agoraphobia and separation anxiety disorder did have a low overall number of cases in this study hampering reliability estimates.For obsessive-compulsive disorder, different criteria for thoughts and behavior revealed divergent responses patterns between both interviews. For obsessive thoughts, mainly the response to the A criterion changed between both interviews. For compulsive behavior, the list of items/behaviors was expanded in the DIA-X-5 to also probe for OCD-related syndromes including nail biting, hair pulling, skin picking, and mirror checking. Although the mere presence of these symptoms was not counted toward the standard diagnosis of OCD, their inclusion may have affected the responses for OCD, given that symptoms such as nail biting were quite prevalent in the sample. Most discordant OCD-cases were due to the B criterion – referring to distress/impairment (mostly rated “some” in the second interview instead of “much”).As already noted, the stem items of most diagnoses showed high reliability, even for the disorders for which relatively low reliability indices were found on the diagnostic level. It should be noted though that the stem question for the use of legal drugs, i.e. medication, reveals low reliability indices. This might depend on the type of listed substances and the broad open category of “other medications”. Unfortunately, no cases of medication use disorder were found in the current study, not allowing to test whether retest-reliability for medication would be higher on the diagnostic level.When comparing the retest-reliability of diagnoses and stem items of the DIA-X-5 with previous results of the DIA-X/M-CIDI , kappa vHigh test-retest reliability was found for the age of onset and age of recency questions in the DIA-X-5; the persistence questions generally revealed slightly lower reliability. Compared to the previous DIA-X/M-CIDI, the ICCs for age of onset in the DIA-X-5 were either similar or somewhat higher for most disorders. Substantially higher ICCs for age of onset were found in the DIA-X/M-CIDI, however, for most specific phobia subtypes, which could be due to the greater number of specific phobia cases in the DIA-X/M-CIDI retest study. In the current study low ICC in age of onset resulted from an overall small number of cases. For the specific phobia subtype, this was combined with an outlier who reported extremely different age of onsets in both interviews. Low ICC’s for age of recency in disruptive mood dysregulation disorder also may be due to few overall cases.Persistence revealed a stronger variability than the other two time related measures. For specific phobias , participants reported diverging onset/recency, most likely because these disorders often manifest early in development and take a waxing and waning course. However, overall persistence - meaning the overall number of years affected - is remembered similarly in both interviews. For illness anxiety and somatic symptom disorder a slow development and varying intensity levels can be assumed leading to difficulties in estimating persistence in terms of number of years affected. For separation anxiety disorder there were too few cases in this study to make reliable conclusions.less frequently diagnosed in the sample. However, previous studies on retest reliability of structured clinical interviews included samples of comparable and even lower size with a range of 60 to 43 participants for the M-CIDI [This test-retest study has limitations: First, the number of subjects assessed is at the lower bound for a test-retest reliability study, which principally affects the reliability estimates for conditions e M-CIDI ) and thee M-CIDI ). Seconde M-CIDI , those aThe DIA-X-5 is an extended, modified version of the DIA-X/M-CIDI. For the mental disorders with sufficient case numbers and thus analyzed in the present study, the DIA-X-5 reliably assesses symptoms, syndromes and diagnoses according to DSM-5 including their onset, recency, and persistence, when applied face-to-face by trained interviewers. These disorders are from DSM-5 sections containing the most prevalent mental disorders: depressive, anxiety, trauma-related, somatic symptom, eating, substance use disorders, and disruptive, impulse control or conduct disorders. The limited sample size calls for additional studies replicating the findings and allowing for more reliable conclusions with regard to less prevalent disorders. Finally, this study focused on the reliability of the DIA-X-5. However, its validity needs to be evaluated in a separate study.


Using these mixed-mode WAX microspheres as packing material, a reliable and robust analytical method based on solid phase extraction and high performance liquid chromatography with ultraviolet detection (SPE-HPLC-UV) was developed for simultaneous determination of six strongly and weakly acidic nonsteroidal anti-inflammatory drugs  in reservoir water. Under optimized conditions, it was applicable to preconcentrate up to 500 mL of reservoir water samples on the WAX cartridges with satisfying recoveries (88–96%) for all the NSAIDs tested. The limits of detection were in the range of 0.002–0.025 μg L−1, respectively. Our results showed that the developed mixed-mode WAX poly(DEAEMA-co-DVB) phase containing a tertiary amine with a pKa value of approximately 10.7 could be used for simultaneous clean-up and preconcentration of strongly and weakly acidic organic pollutants in real environmental water, which could not be achieved by single use of quaternary ammonium strong anion-exchange phase or weaker primary and secondary amine anion-exchange.Poly(2-(diethylamino)ethyl methacrylate-co-divinylbenzene) (poly(DEAEMA-co-DVB)) microspheres with mixed-mode weak anion-exchange (WAX) character were successfully fabricated for the first time via facile one-step Pickering emulsion polymerization. The obtained poly(DEAEMA-co-DVB) particles had good spherical geometry, uniform particle size in the range of 30–40 µm, a large specific surface area of 575 m Despite those great progresses achieved in improving precision and accuracy of analytical instruments, determination of analytes at trace level in complex matrices is still a huge challenge for direct instrumental analysis. At this time, the pretreatment step with the purpose of separation and/or enrichment of target analytes becomes a key prerequisite for accurate analysis. Even for high-sensitivity analyses, such as those employing liquid chromatograph-mass spectrometer/mass spectrometer (LC-MS/MS), proper sample preparation can be critical for minimizing matrix effects and concentrating analytes of interest ,2. AmongThe principle of solid phase extraction is that the target analyte can be retained on the adsorbent by the interaction between them with the aim of enrichment and separation. According to the mode of interaction, the adsorbent can be roughly classified into two types, a single-mode adsorbent, and a mixed adsorbent ,9. Singl−1 to μg L−1. The presence of NSAIDs residues in natural water systems has raised major concerns for their potential risk to public health. Most NSAIDs contain a carboxylic acid group that can exist in protonated  state or in an ionized (charged) form. Therefore, mixed-mode SAX (SAX) sorbent such as Oasis MAX (pKa > 18) perform well in retaining and releasing weakly acidic NSAIDs at their deprotonation and protonation states, respectively. However, niflumic acid is a relatively strong acid (pKa = 1.68) that maintains ionization over the whole pH range investigated. As a result, mixed-mode SAX can be challenging as it is difficult to disrupt the ion-exchange interaction by only pH adjustment. Mixed-mode weak anion-exchange (WAX) sorbent with primary, secondary, and tertiary amine as functional groups introduces additional selectivity, which allows users to control the ionization of stationary phase by modifying the pH. In conclusion, mixed-mode SAX is for weakly acidic analytes while mixed-mode WAX is for strongly acidic analytes, since irreversible binding happens between a strong analyte and strong sorbent and it is difficult for weak sorbent to attract a weak analyte.Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most frequently used pain killer medicines, and NSAIDs cause toxicity in the environment even at very low concentration of ng LCompared to silica-based sorbents, polymer-based sorbents have wide pH range and no silanol interactions exists . The OasIn current work, we used a facile one-step Pickering emulsion polymerization for preparing poly(DEAEMA-co-DVB) microspheres that could simultaneously and efficiently extract strongly (niflumic acid) and weakly  acidic NSAIDs. The developed poly(DEAEMA-co-DVB) sorbent contains a tertiary amine with a pKa value of around 10.7, which allows for control of the ionization of stationary phase by modifying the pH. Tertiary amine functionality can easily exchange strongly acidic NSAIDs anion. Furthermore, tertiary amine is more basic than primary amine (pKa ≈ 6), imidazole and pyridine, and therefore provides better anion-exchange (acid-base) interactions with those weak acidic NSAIDs. Therefore, our poly(DEAEMA-co-DVB) WAX can simultaneously enrich, purify, and analyze strongly and weakly acidic NSAIDs.2 (12 nm particle diameter) supplied by Sigma-Aldrich , and 2-(diethylamino)ethyl methacrylate  as well as n-dodecanol (99.0%) from Tokyo Chemical Industry Co. . The pharmaceutical analytes were naproxen  from J&K Scientific , ketoprofen  from Sigma-Aldrich , and niflumic acid , diflunisal , mefenamic acid , and diclofenac  obtained from Accela Chem Bio . To remove inhibitors from DVB and DEAEMA, they were treated by passing through short columns containing 2 g of alumina-B . The water used in all experiments was deionized water from a Milli-Q purification system . The empty polypropylene SPE tubes (3 mL) with bio-fiber frits were supplied by Shenzhen Biocomma Technologies Co. .The reagents involved for synthesizing the polymer were divinylbenzene (DVB) and 2,2-azobisiobutyronitrile (AIBN) supplied by J&K Scientific , nano-SiOThe mixed-mode WAX microspheres were synthesized via one-step Pickering emulsion polymerization procedure. First, 0.422 g of n-dodecanol, 22.5 mg of AIBN, 1.2 mL of toluene, 0.4 mL of DEAEMA, and 1.2 mL of DVB were mixed, followed by the treatment of nitrogen purge. Then, 120 mg of silica and 10 mL of water were mixed and vortexed for 1 min. Next, the mixed precursor solution was added to this solution, uniformly dispersed with a high-speed disperser, and placed in a sealed tube. The polymerization was carried out at 60 °C for 24 h. After polymerization, the polymer was washed with ethanol and dried. The solid was soaked in hydrofluoric acid for 24 h to etch away the silica, and then extracted with Soxhlet extraction for 12 h. The withdrawn material was drained, and a new WAX sorbent was obtained in this way.Water samples were collected from Xishan Reservoir , part of the city water supply system, stored on ice and immediately transported to the laboratory. Then, the samples were filtered through 0.45 μm fibers to remove particulate matter, stored in clean glass bottles and kept in a refrigerator at 4 °C until SPE process.2O. After conditioning, the water sample was poured into the cartridge. The cartridge was first washed with 2 mL of MeOH/H2O (1:1), and then the target analytes were eluted out using 2 mL of NH3·H2O/MeOH (1:49). The extract was evaporated to dryness under nitrogen, reconstituted in 1 mL of the initial mobile phase and then filtered for HPLC-UV analysis.The obtained materials were characterized by the following analytical techniques. Scanning electron microscopy (SEM) was performed on a JSM-7800F scanning electron microscope . Nitrogen adsorption–desorption isotherms were performed at 77 K using a quantachrome Autosorb-iQ2 instrument . An approximately 0.1-g sample was degassed under vacuum at 80 °C for 12 h prior to measurement. The FT-IR spectra were 50 mg of poly(DEAEMA-co-DVB) was packed into 3 mL SPE tube. The tube was first conditioned with 3 mL of MeOH and 3 mL of HThe chromatographic system was an Agilent 1200 series HPLC system  equipped with a quaternary pump, an on-line degasser, an autosampler and a diode array detector (DAD). Chromatographic separations were performed on an Agilent ZORBAX Eclipse XDB-C18 column . The mobile phase was composed of Solvent A (methanol) and Solvent B (20 mM ammonium acetate). The chromatographic separation was run as follows: a gradient from 30% to 75% of Solvent A in 20 min, then returned to initial condition in 0.5 min, and held for 3.5 min. All sample injection volumes were 20 μL. The column temperature was kept at 25 °C. The detection wavelengths were set at 230 nm for NIF, and 288 nm for NAP, KEP, DIF, MEF, DIC.2-stabilized Pickering emulsion polymerization method. By self-organization of SiO2 at the interface of oil (monomers and porogens) and water, the stabilized oil-in-water Pickering emulsion droplets were formed and used as polymerization vessels to obtain the polymer/SiO2 composites by thermal radical polymerization. The SiO2-covered spherical particles were then etched using hydrofluoric acid to remove SiO2, and the poly(DEAEMA-co-DVB) microspheres were finally obtained. Here, the monomers DEAEMA and DVB afforded the weak anion-exchange and hydrophobic functionalities, respectively, and a combination of them successfully constructed the mixed-mode weak anion-exchange medium.As shown in A dose of 120 mg for silica nanoparticles was adopted with the aim of optimized uniformity and particle size. The synthesized solid material was subsequently characterized by SEM, nitrogen physical absorption, and FT-IR measurements. The SEM results shown in 2 sorption–desorption isotherm gives the porous structure of poly(DEAEMA-co-DVB), as shown in The N2/g and 0.94 cm3/g, respectively, which is very beneficial for the adsorption of pharmaceutical molecules in solution. Additionally, the FT-IR was also carried out for identify the functional groups, and the results are given in −1 is ascribed as the stretching vibration of tertiary amine in poly(DEAEMA-co-DVB). The DVB as the copolymerized monomer is approved by the peaks at 1604 and 1510 cm−1 attributed to C=C, and the peaks at 796 and 710 cm−1 are attributed to =C–H of benzene ring. Moreover, the signal at 1727 cm−1 is testified as the C=O stretching of ester, which is different from the fact that carbonyl signal of –COOC=C appears in DEAEMA monomer, strongly implying that the vinyl-copolymerization process of DEAEMA and DVB was successful due to the disappearance of vinyl group. In addition, the existence of 1205 and 1118 cm−1 for C–O–C stretching was also assigned to the ester group. Thus, the IR test confirms the correct synthesis of poly(DEAEMA-co-DVB) material.As presented in We chose a group of acidic pharmaceuticals ranging from strongly acid to weak ones as target analytes , and their molecular structures, log P, and pKa values are listed in 2O/MeOH (1:1). In the pH range around pKa, small changes in pH provide major changes in ionization and retention. Higher pH above their pKa values leads to an increase in the degree of ionization of acidic targets, making the acidic drug mainly retained by ion-exchange mechanism. As the sample pH decreases, the degree of ionization of the acidic drug also decreases, but at this time the acidic target can be well retained on the column by hydrophobic interaction. In conclusion, in the entire pH range, the two complement interaction mechanisms can retain the acidic analytes well, in whatever form they exist. This was confirmed by the fact that the recoveries of all analytes varied little with the changes in the investigated pH range. As shown in Before conducting the experiment, we selected the sample pH of 1, 3, 5, and 7 for investigating its effect on recoveries of all pharmaceuticals. After loading the sample, the cartridges were washed with 4 mL of H3·H2O was selected as the elution solvent to deprotonate the stationary phase. Theoretically, the amount of elution solvent is positively related to the elution ability; however, to minimize the volume of the elution solution and reduce the nitrogen purge time for subsequent experiment, the optimization experiments of elution volume, including 1 × 2, 1.5 × 2, 2 × 2, and 2.5 × 2 mL, were carried out. The experimental results are shown in To effectively elute an analyte that is being retained on a mixed-mode surface via two retention mechanisms, it is necessary to use an elution solvent that simultaneously disrupts both of the retention mechanisms. Therefore, methanol with 2% NHLarge volume water analysis is beneficial for a larger preconcentration factor and thus higher detection sensitivity. To determine the sample breakthrough volume, 50, 100, 200, 500, and 1000 mL volumes of ultrapure water containing 0.5 μg/L of NAP and 5 μg/L of KEP, DIF, MEF, DIC, and NIF were percolated through the column. The results in −1 for NAP and 0.05–10.0 μg L−1 for KEP, DIF, MEF, DIC, and NIF with the correlation coefficients (r2) > 0.994. Using signal to noise method, the estimated LOD and LOQ were in the range of 0.002–0.025 and 0.003–0.088 μg L−1, respectively, as shown in n = 3) of the recoveries obtained by analyzing 500 mL of spiked samples within one day and during a period of three consecutive days, respectively. Moreover, the outstanding repeatability of good intra- and inter-day shown in The signal intensity (peak area) for each target analyte is proportional to its concentration within the range of 0.01–2.0 μg LWe applied the established method for analysis of real environmental water samples. Our analysis results show that the six targets were not detectable in the reservoir water using the current established method. To test the applicability of our optimized method to real samples, spiked reservoir samples were used instead. As shown in For most pharmaceuticals, our results were comparable or even superior to Oasis MAX, especially for NIF. NIF is a strong acid that remains ionized across the whole pH range. Thus, NIF has a very strong interaction with the SAX Oasis MAX so that it is not possible to disrupt the ion exchange mechanism by pH adjustment, thus resulting in a low recovery of 30%. However, our WAX SPE utilized a hydrophobic backbone functionalized with a tertiary amine with a pKa of approximately 10.6, which allows easy control of the ionization of the stationary phase by modifying the pH. At the same time, a pKa of approximately 10.6 guaranteed that the stationary phase was positively charged across the investigated pH range. As a result, our WAX SPE can be applicable to both strongly and weakly acidic compounds. It was demonstrated that the laboratory-made poly(DVB-co-DEAEMA) can be applied to the extraction of NSAIDs from NIF (strongly acidic) to KEP, DIF, MEF, DIC, and NIF (weakly acidic), and the detection limit can be as low as 0.01 μg/L. In addition, our results are comparable to the performance of those mixed-mode SPE sorbents used in other previous reports. In conclusion, the laboratory-made poly(DVB-co-DEAEMA) can be an excellent mixed-mode SPE material for sample pretreatment of acidic organic pollutants in environmental samples.For the first time, a mixed-mode WAX poly(DVB-co-DEAEMA) utilizing a hydrophobic backbone functionalized with a tertiary amine (with a pKa of approximately 10.6) was successfully used for SPE extraction of widely acidic pharmaceuticals  from real water samples. This result was difficult to achieve by mixed-mode SAX and mixed-mode WAX containing weaker primary and secondary amines. The former cannot perform well for strong acids while the latter was not a good choice for weakly acidic analytes. In contrast to both, our mixed-mode WAX SPE can be successfully applied in simultaneous efficient enrichment of both strongly and weakly acidic NSAIDs with satisfying recoveries for the first time. The other importance of this work lies in that the functionalized sorbent was synthesized in a well-defined micro-sphere format by simple one-step Pickering emulsion polymerization. The method is process-simplified, as only one step was included in the fabrication. Finally, the application of our mixed-mode WAX not only allows selective extraction of the target analytes but also cleans up the sample, which are important particularly when the sample is complex and impurities can interfere with quantification.